Phrodo green am intracellular ph indicator

Manufactured by Thermo Fisher Scientific

Sourced in United States

The PHrodo Green AM Intracellular pH Indicator is a fluorescent dye used to measure intracellular pH levels. It is a cell-permeant indicator that can be loaded into cells and emit fluorescence in response to changes in pH within the cellular environment.

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Lab products found in correlation

Micro ph electrode, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle medium f12, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Accutase cell detachment solution, by Merck Group (1 mentions) Powerload concentrate, by Thermo Fisher Scientific (1 mentions) Intracellular ph calibration kit, by Thermo Fisher Scientific (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Ab15 basic, by Thermo Fisher Scientific (1 mentions) Ph calibration buffer kit, by Thermo Fisher Scientific (1 mentions) Facsverse cytometer, by BD (1 mentions) Antimycin a, by Agilent Technologies (1 mentions) Hepes, by Carl Roth (1 mentions) Rotenone, by Agilent Technologies (1 mentions) Axiovert, by Zeiss (1 mentions) Infinite m200, by Tecan (1 mentions) Bz x710, by Keyence (1 mentions) Lsm 700 confocal microscope, by Zeiss (1 mentions) Zen software, by Zeiss (1 mentions) Victor nivo, by PerkinElmer (1 mentions)

Close spelling variants of this product

LysoSensor pHrodo Green AM intracellular pH indicator PHrodo Green Intracellular pH Indicator PHrodo Green AM PHrodo green

16 protocols using phrodo green am intracellular ph indicator

ARPE-19 Cell Culture and pH Measurement

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ARPE-19 cells were cultured in complete Dulbecco’s Modified Eagle Medium F12 (Gibco Life Technology, Gaithersburg MD; DMEM F12) medium containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (100-units penicillin/ml, 100 μg streptomycin/ml), (Invitrogen, San Diego, California, USA). Cells were grown in an incubator at 37°C with 5% CO₂ using six-well plates and culture chambers (Corning Primaria plastic culture ware). The culture plates used in these experiments were not coated with collagen because preliminary findings showed cells cultured in collagen coated wells showed no difference in amelotin expression by in situ hybridization and immunofluorescence (Fig. S1) to cells cultured directly on plastic. Cells were grown to a minimum of 90% confluence prior to serum deprivation experiments.
The extracellular pH of the confluent cells cultured with either serum deprived media or 10% serum supplemented media were measured by slightly tilting the six-well culture plates to pool the media and inserting a micro pH electrode (Thermo Fisher Scientific, Waltham, MA). The pH reading was performed once a day from two replicate wells for each culture condition for nine days. Intracellular pH of ARPE-19 cells cultured in 10% serum or SFM were labeled with pHrodo Green AM Intracellular pH Indicator (Thermo Fisher Scientific, Waltham, MA) following manufacture’s protocol.

RAJAPAKSE D., PETERSON K., MISHRA S., FAN J., LERNER J., CAMPOS M, & WISTOW G. (2020). Amelotin is expressed in retinal pigment epithelium and localizes to hydroxyapatite deposits in dry age-related macular degeneration. Translational research : the journal of laboratory and clinical medicine, 219, 45-62.

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Cytosolic pH Measurement in MCF-7 Cells

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To estimate the cytosolic pH, MCF-7 cells were washed with RPMI without phenol red and then incubated for 30 min at 37 °C in the same solution, containing 1:1000 dilution of pHrodo™ Green AM Intracellular pH Indicator and 1:100 dilution of PowerLoad™ concentrate (Thermo Fisher Scientific). Next, the cells were washed with RPMI without phenol red, and 2 h incubation with and without LNCs (0.5 and 1 mg/ml) was performed under cell growth conditions, in a medium supplemented with 1 or 10% FCS. Subsequently, the cells were harvested by Accutase^® Cell Detachment Solution (Sigma-Aldrich, St Louis, MO), centrifuged (300×g for 10 min at 4 °C), washed and resuspended in PBS. A standard curve was established by incubating cells as described above, and after detachment from wells, for 5 min at 37 °C with a series of pH calibration buffers (pH 4.5, 5.5, 6.5, and 7.5) supplemented with 10 µM valinomycin and 10 µM nigericin (Intracellular pH Calibration Kit; Thermo Fisher Scientific). Fluorescence intensity was measured by LSR II Flow Cytometer (BD Biosciences, San Jose, CA), using a 505 nm Ar laser filter and detected with the FL1 530/30 nm bandpass filter. At least 5000 cells were recorded in duplicates.

Szwed M., Torgersen M.L., Kumari R.V., Yadava S.K., Pust S., Iversen T.G., Skotland T., Giri J, & Sandvig K. (2020). Biological response and cytotoxicity induced by lipid nanocapsules. Journal of Nanobiotechnology, 18, 5.

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Intracellular pH Measurement using pHrodo Green

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In the intracellular pH measurements, the pHrodo Green AM intracellular pH indicator from ThermoFisher Scientific was employed. To prepare the dye solution, 10 μL of pHrodo Green AM was combined with 100 μL of PowerLoad concentrate. This resulting mixture was further diluted with 10 mL of PBS. The growth medium was aspirated from the cells, which were subsequently rinsed once with PBS. The pHrodo Green AM/PowerLoad/PBS mixture was introduced to the cells, followed by an incubation period at 37 °C for 30 min to allow for dye uptake. Subsequently, the cells were subjected to analysis using a fluorescence plate reader with excitation at a maximum of 509 nm and emission at a maximum of 533 nm. This measurement enabled the assessment of intracellular pH levels.

Gurhan H, & Barnes F. (2023). Impact of weak radiofrequency and static magnetic fields on key signaling molecules, intracellular pH, membrane potential, and cell growth in HT-1080 fibrosarcoma cells. Scientific Reports, 13, 14223.

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Nanomaterials Impact on Tumor Microenvironment

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To estimate how nanoCaCO₃ were affecting the tumor microenvironment, pH measurements were made using 24 well plates. First, MDA-MB-231 cells were seeded in the wells at a concentration of 3.0 × 10⁶ cells mL⁻¹. The next day, the media was replaced with either plain media (control) or media containing 0.8 mg mL^-1 nanoCaCO₃ (experimental). After 3 more days, the media was collected, and a pH probe (AB15 Basic, ThermoFisher) was used to measure the pH of the media in the wells.
Additionally, the intracellular pH was determined using a pHrodo Green AM Intracellular pH Indicator (ThermoFisher) following the manufacture’s protocol. With this dye, higher fluorescent intensities indicate a lower pH. Therefore, to correlate fluorescent intensities with pH values, a pH Calibration Buffer Kit (ThermoFisher) was used.

Lam S.F., Bishop K.W., Mintz R., Fang L, & Achilefu S. (2021). Calcium carbonate nanoparticles stimulate cancer cell reprogramming to suppress tumor growth and invasion in an organ-on-a-chip system. Scientific Reports, 11, 9246.

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Assessing Enteroid pH Regulation

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Enteroids were differentiated for 5–7 days in the presence of vehicle (water or DMSO), 10 nM VIP (Tocris), 10 nM PYY (Phoenix Pharmaceuticals) and/or 300 nM BIBO3304. On the final day, enteroids were removed from Matrigel and enzymatically dissociated into single-cell suspension using 0.25% Trypsin-EDTA. Cell suspensions were counted and equal cell numbers of dissociated HIOEs were incubated in pHrodo Green AM Intracellular pH indicator (ThermoFisher Scientific) according to manufacturer’s directions for 30 min at 37 C, washed with 1X PBS, and analyzed by flow cytometry.

McCauley H.A., Matthis A.L., Enriquez J.R., Nichol J.T., Sanchez J.G., Stone W.J., Sundaram N., Helmrath M.A., Montrose M.H., Aihara E, & Wells J.M. (2020). Enteroendocrine cells couple nutrient sensing to nutrient absorption by regulating ion transport. Nature Communications, 11, 4791.

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Intracellular pH Measurement of MCF10A Cells

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The pH of cell lines of the MCF10A progression series were measured with the pHrodo® Green AM Intracellular pH Indicator (Cat# P35373, Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, live cells were incubated with the pH probe for 30 min. For standard samples, the standard solution (Cat# P35379, Thermo Fisher Scientific) for each different pH was added at the end of 30 min, and cells were incubated for another 5 min. Cells were washed in fresh media, and micrographs were taken on live cells by fluorescent microscope with FITC filter. The signal intensity per cell was quantified by ImageJ.

Ren G., Zheng X., Bommarito M., Metzger S., Walia Y., Letson J., Schroering A., Kalinoski A., Weaver D., Figy C., Yeung K, & Furuta S. (2019). Reduced Basal Nitric Oxide Production Induces Precancerous Mammary Lesions via ERBB2 and TGFβ. Scientific Reports, 9, 6688.

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Intracellular pH Measurement Using pHrodo™

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The intracellular pH calibration buffer kit was used to measure the intracellular pH of live cells in conjunction with the pHrodo™ green AM intracellular pH indicator (a fluorogenic probe) according to the manufacturer’s protocol (Thermo Fisher Scientific).⁶⁴ Briefly, the cells were washed with live cell imaging solution (LCIS). Ten microlitres of pHrodo™ Green AM was mixed with 100 μL of PowerLoad™ and added to 10 mL of LCIS. The cells were incubated with this mixture for 30 min at 37 °C, followed by pH measurement using a spectrometer. The cells were treated with lactic acid, phloretin or lonidamine, as required, in DMEM without phenol red. The fluorescence intensity of the cells was measured by spectrometer screening to analyze the pH of the cells.

Zhang J.Y., Zhou B., Sun R.Y., Ai Y.L., Cheng K., Li F.N., Wang B.R., Liu F.J., Jiang Z.H., Wang W.J., Zhou D., Chen H.Z, & Wu Q. (2021). The metabolite α-KG induces GSDMC-dependent pyroptosis through death receptor 6-activated caspase-8. Cell Research, 31(9), 980-997.

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Measuring Intracellular pH in E. coli

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pHrodo® Green AM Intracellular pH Indicator (Thermo Fisher) was used to measure intracellular pH in live E. coli. Cells were first washed with live-cell imaging solution (LCIS) and then 10 μl of pHrodo^™ Green AM with 100 μl of PowerLoad^™ concentrate were added to 10 ml of LCIS. The pHrodo^™ AM/PowerLoad^™/LCIS was mixed with cells and incubated at 37°C for 30 minutes. Cells were then washed twice with PBS to remove excess dye before flow-cytometric analysis. Intracellular pH calibration buffers (Thermo Fisher) were used as standards.

Xia J., Chiu L.Y., Nehring R.B., Núñez M.A., Mei Q., Perez M., Zhai Y., Fitzgerald D.M., Pribis J.P., Wang Y., Hu C.W., Powell R.T., LaBonte S.A., Jalali A., Guzmán M.L., Lentzsch A.M., Szafran A.T., Joshi M.C., Richters M., Gibson J.L., Frisch R.L., Hastings P.J., Bates D., Queitsch C., Hilsenbeck S.G., Coarfa C., Hu J.C., Siegele D.A., Scott K.L., Liang H., Mancini M.A., Herman C., Miller K.M, & Rosenberg S.M. (2019). Bacteria-to-human protein networks reveal origins of endogenous DNA damage. Cell, 176(1-2), 127-143.e24.

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Intracellular pH Measurement Methodology

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Extracellular medium pH was assessed using a pH meter (pH 211 Microprocessor pH Meter with pH electrode HI1131B; both Hanna instruments, Vöhringen, Germany) and calibration solutions (Buffer solution pH 4.0 and pH 7.0; both Honeywell, Morristown, USA).
Intracellular acidification was measured using pHrodo Green AM Intracellular pH Indicator (Thermo Fisher Scientific Inc., Waltham, USA) as indicated by the manufacture. Briefly, pHrodo dye was added 1:10 into PowerLoad concentrate and this mixture then was further diluted 1:100 in Live Cell Imaging Solution (140mM NaCl, 2.5mM potassium chloride, 1.8mM calcium chloride, 1mM magnesium chloride (all from Merck KGaA, Darmstadt, Germany), and 20mM HEPES ((Carl Roth, Karlsruhe, Germany); pH 7.4). Growth medium was replaced by this staining solution and cells were incubated at 37°C for 30 minutes (min) prior to measurement. This was done either directly using cells from culture or after one-hour priming with Na-L-lactate and six hours stimulation with LPS. As a positive control, the respiratory chain was inhibited by antimycin A and rotenone (both Agilent, Santa Clara, USA). A FACSVerse cytometer (BD Biosciences, Franklin Lakes, USA) was used for measurement. Results were calculated using mean fluorescent intensity.

Schenz J., Heilig L., Lohse T., Tichy L., Bomans K., Büttner M., Weigand M.A, & Uhle F. (2021). Extracellular Lactate Acts as a Metabolic Checkpoint and Shapes Monocyte Function Time Dependently. Frontiers in Immunology, 12, 729209.

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Acidic Vesicle Detection via pHrodo

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Detection of acidic environments of vesicles was accomplished utilizing pHrodo Green AM Intracellular pH indicator (ThermoFisher Scientific). Cells were grown in phenol-red-free DMEM media (for SILAC) (ThermoFisher) supplemented with 2% fetal bovine serum, 3151 mg/L glucose, 147.5 mg/L L-arginine HCL, and 91.25 mg/L L-lysine HCL. Prior to laser exposure, cells were incubated for 30 minutes in 5 μM pHrodo with 1000x powerload concentrate at 37°C. Fluorescence imaging was performed on a Zeiss Axiovert using a GFP filter set.

Wakida N.M., Cruz G.M., Ro C.C., Moncada E.G., Khatibzadeh N., Flanagan L.A, & Berns M.W. (2018). Phagocytic response of astrocytes to damaged neighboring cells. PLoS ONE, 13(4), e0196153.

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